Metabonomics analysis of semen euphorbiae and semen Euphorbiae Pulveratum using UPLC–Q-TOF/MS

Semen Euphorbiae (SE), the dry and mature seed of Euphorbia lathyris L., a common traditional Chinese medicine, has significant pharmacological activity. However, its toxicity limits its clinical application, and less toxic Semen Euphorbiae Pulveratum (SEP) is often used clinically. To explore the possible mechanism of SE frost-making and attenuation, this study used ultrahigh-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry to perform a comprehensive metabolomics analysis of serum and urine samples from rats treated with SE and SEP, and performed histopathological evaluation of liver, kidney and colon tissues. Meanwhile, the different metabolites were visualized through multivariate statistical analysis and the HMDB and KEGG databases were used to distinguish the differential metabolites of SE and SEP to reveal related metabolic pathways and their significance. In total, 32 potential biomarkers, 14 in serum and 18 in urine, were identified. The metabolic pathway analysis revealed that arachidonic acid metabolism, sphingolipid metabolism, tyrosine and tryptophan biosynthesis, the tricarboxylic acid cycle and seven other metabolic pathways were significantly altered. Importantly, compared with SE, SEP reduced the metabolic disorder related to endogenous components. The mechanism may be related to the regulation of lipid metabolism, intestinal flora metabolites, amino acid metabolism and energy metabolism. This study provided new insights into the possible mechanism of SE freezing and attenuation.     

A fast,sensitive, and high‐throughput method for the simultaneous quantitation of three ellagitannins from Euphorbiae pekinensis Radix in rat plasma by ultra‐HPLC–MS/MS

A fast, sensitive, and high‐throughput ultra‐HPLC–MS/MS method has been developed and validated for the simultaneous determination of three main active constituents of Euphorbiae pekinensis Radix in rat plasma. After addition of the internal standard, plasma samples were extracted by liquid–liquid extraction with ethyl acetate/isopropanol (1:1, v/v) and separated on a CAPCELL PAK C₁₈ column (100 × 2.0 mm, 2 μm, Shiseido, Japan), using a gradient mobile phase system of methanol/water. The detection of the analytes was performed on a 4000Q UHPLC–MS/MS system with turbo ion spray source in the negative ion and multiple reaction‐monitoring mode. The linear range was 1.0–1000 ng/mL for 3,3′‐di‐O‐methyl ellagic acid‐4′‐O‐β‐d ‐glucopyranoside (i), 1.5–1500 ng/mL for 3,3′‐di‐O‐methyl ellagic acid‐4′‐O‐β‐d ‐xylopyranoside (ii), and 5.0–5000 ng/mL for 3,3′‐di‐O‐methyl ellagic acid (iii). The intra‐ and interday precision and accuracy of all the analytes were within 15%. The extraction recoveries of the three analytes and internal standard from plasma were all more than 80%. The validated method was first successfully applied to the evaluation of pharmacokinetic parameters of compounds 1 , 2 , and 3 in rat plasma after intragastric administration of the Euphorbiae pekinensis Radix extract.     

Simultaneous Determination of Lathyrane Diterpenoids by NPLC-DAD in the Seeds of Euphorbia lathyris and “ZI-JIN-DING” Pastille

A novel, simple and specific normal-phase liquid chromatography (NPLC) method has been developed for simultaneous determination of the four lathyrane diterpenoids, lathyrol-5,15-diacetate-3-benzoate (1, L₃), lathyrol-5,15-diacetate-3,7-dibenzoate (2, L₂), Δ6,17-epoxide-5,15-diacetate-3-phenylacetate (3, L₁), lathyrol-5,15-diacetate-3-nicotinate (4, L₈) in the hexane extract of the seeds of Euphorbia lathyris and “ZI-JIN-DING” pastille. The method showed good linearity for the four analytes (r ² > 0.99), the intra-day and inter-day variations (RSD) were less than 3%, and the recoveries ranged from 99.4 to 100.6%. This method was successfully used to determine the four lathyrane diterpenoids in the seeds of Euphorbia lathyris and “ZI-JIN-DING” pastille, and could be applied for their quality control.     

Determination of 12 potential nephrotoxicity biomarkers in rat serum and urine by liquid chromatography with mass spectrometry and its application to renal failure induced by Semen Strychni

In previous nephrotoxicity metabonomic studies, several potential biomarkers were found and evaluated. To investigate the relationship between the nephrotoxicity biomarkers and the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni‐induced renal failure, 12 typical biomarkers are selected and a simple LC–MS method has been developed and validated. Citric acid, guanidinosuccinic acid, taurine, guanidinoacetic acid, uric acid, creatinine, hippuric acid, xanthurenic acid, kynurenic acid, 3‐indoxyl sulfate, indole‐3‐acetic acid, and phenaceturic acid were separated by a Phenomenex Luna C₁₈ column and a methanol/water (5 mM ammonium acetate) gradient program with a runtime of 20 min. The prepared calibration curves showed good linearity with regression coefficients all above 0.9913. The absolute recoveries of analytes from serum and urine were all more than 70.4%. With the developed method, analytes were successfully determined in serum and urine samples within 52 days. Results showed that guanidinosuccinic acid, guanidinoacetic acid, 3‐indoxyl sulfate, and indole‐3‐acetic acid (only in urine) were more sensitive than the conventional renal function markers in evaluating the therapeutic role of Radix Glycyrrhizae extract on Semen Strychni‐induced renal failure. The method could be further used in predicting and monitoring renal failure cause by other reasons in the following researches.     

Isolation and determination of p-hydroxybenzoylcholine in traditional Chinese medicine Semen sinapis Albae

A special component is isolated from Semen sinapis Albae (white mustard seed), a traditional Chinese medicine. According to the physical and chemical investigation and spectroscopic identification, this component can be known as p-hydroxybenzoylcholine bisulfate, a choline base. This component in the drug is also determined by RP-HPLC. A reversed-phase C₁₈ column is used to separate the p-hydroxybenzoylcholine with an eluent of methanol–0.05 mol/L monopotassium phosphate solution (30:70) (adjusted by phosphoric acid to pH 3.6) at the flow rate of 0.5 mL/min. Detection is carried out with a UV detector operated at 285 nm, and the column temperature is 25 °C. It reveals that there is 0.021% (w/w) of p-hydroxybenzoylcholine bisulfate in Semen sinapis Albae and 0.037% (w/w) in stir-baked Semen sinapis Albae.     

The metabolites and biotransformation pathways in vivo after oral administration of ocotillol,RT5, and PF11

Ocotillol, pseudo-ginsenoside RT5 (RT₅), and pseudo-ginsenoside F₁₁ (PF₁₁) are ocotillol-type saponins that have the same aglycone structure but with different numbers of glucose at the C-6 position. In this study, the metabolites of ocotillol, RT₅, and PF₁₁ in rat plasma, stomach, intestine, urine, and feces after oral administration were investigated by ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry. The results showed that RT₅ was easily biotransformed into metabolites in vivo, whereas PF₁₁ and RT₅ were difficult to be biotransformed. Hydrogenation, dehydrogenation, dehydration, deglycosylation, deoxygenation, hydration, phosphorylation, deoxidation, glucuronidation, and reactions combining amino acid were speculated to be involved in the biotransformation of ocotillol, RT₅, and PF₁₁. Based on the structural analysis of metabolites, it was deduced that hydrogenation, dehydration, deoxidation, and reactions combining amino acid occurred on the aglycone structure, whereas deglycosylation, hydration, and phosphorylation occurred on the glycosyl chain. Further, metabolites in plasma, urine, feces, and tissues were different: First, glucuronidation products were found in urine, stomach, intestine, and feces, but not in plasma. Second, the ocotillol prototype was not identified in urine samples. Third, the RT₅ prototype was found in stomach, intestine, feces, and urine, but not in plasma.     

Anti-Malignant Ascites Effect of Total Diterpenoids from Euphorbiae ebracteolatae Radix Is Attributable to Alterations of Aquaporins via Inhibiting PKC Activity in the Kidney

This study evaluated the anti-ascites effect of total diterpenoids extracted from Euphorbiae ebracteolatae Radix (TDEE) on malignant ascitic mice and elucidated its underlying mechanism. TDEE was extracted by dichloromethane and subjected to column chromatography. The purity of six diterpenoids isolated from TDEE was determined to be 77.18% by HPLC. TDEE (3 and 0.6 g raw herbs/kg, p.o.) reduced ascites and increased urine output. Meanwhile, analysis of tumor cell viability, cycle and apoptosis indicated that TDEE had no antitumor activity. In addition, the expression levels of aquaporins (AQPs) and the membrane translocation levels of protein kinase C (PKC) α and PKCβ in kidney and cells were measured. TDEE reduced the levels of AQP1–4, and inhibited PKCβ expression in membrane fraction. Four main diterpenoids, except compound 2, reduced AQP1 level in human kidney-2 cells. Compounds 4 and 5 inhibited AQP2–4 expression in murine inner medullary collecting duct cells. The diterpenoid-induced inhibition of AQP1–4 expression was blocked by phorbol-12-myristate-13-acetate (PMA; agonist of PKC). The diterpenoids from TDEE are the main anti-ascites components. The anti-ascites effect of diterpenoids may be associated with alterations in AQPs in the kidneys to promote diuresis. The inhibition of AQP1–4 expression by TDEE is related to the inhibition of PKCβ activation.     

Quantitative analysis of biomarkers of liver and kidney injury in serum and urine using ultra‐fast liquid chromatography with tandem mass spectrometry coupled with a hydrophilic interaction chromatography column: Application to monitor injury induced by Euphorbiae pekinensis Radix

Fast and sensitive monitoring of drug‐induced liver and kidney injury in early stage is beneficial. An ultrafast liquid chromatography with tandem mass spectrometry assay was developed and validated to simultaneously determine ten endogenous biomarkers in serum and urine, including hippuric acid, phenylacetylglycine, 5‐oxoproline, cholic acid, taurine, indoleacetic acid, 3‐indoxyl sulfate, guanidinosuccinic acid, guanidinoacetic acid and uric acid. A CAPCELL CORE PC column (2.1 × 150 mm, 2.7 μm) was adopted for analytes separation. Gradient elution was performed with acetonitrile and water containing 5 mM ammonium acetate. Simple protein precipitation was applied in sample preparation. Good linearities were achieved with all the regression coefficients above 0.9911. Accuracy was 2.9–14.2% in serum and 4.1–14.6% in urine. The mean recovery was above 70% with acceptable matrix effects. The method was applied to monitor injury induced by Euphorbiae pekinensis Radix with a subacute rats model. All the biomarkers showed obvious concentration changes during the injury period. Furthermore, several biomarkers showed significant changes in earlier stage when compared with the current clinical serum bio‐parameters. The method might be helpful for early diagnosis of drug induced liver and kidney injury in clinical after tested on more drugs.     

Comprehensive metabolite profiling of Plantaginis Semen using ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique

Plantaginis Semen is commonly used in traditional medicine to treat edema, hypertension, and diabetes. The commercially available Plantaginis Semen in China mainly comes from three species. To clarify the chemical composition and distinct different species of Plantaginis Semen, we established a metabolite profiling method based on ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry coupled with elevated energy technique. A total of 108 compounds, including phenylethanoid glycosides, flavonoids, guanidine derivatives, terpenoids, organic acids, and fatty acids, were identified from Plantago asiatica L., P. depressa Willd., and P. major L. Results showed significant differences in chemical components among the three species, particularly flavonoids. This study is the first to provide a comprehensive chemical profile of Plantaginis Semen, which could be involved into the quality control, medication guide, and developing new drug of Plantago seeds.     

沙苑子提取液对不同体系中草酸钙晶体生长影响的研究

通过与水、氯化钠、正常人尿液体系的比较,重点研究了结石患者尿液体系中加入中药沙苑子提取液对草酸钙晶体生长的的影响,利用SEM,FTIR和XRD等测试手段对所得晶体进行表征。结果发现:在结石患者尿液体系中形成的草酸钙晶体为一水草酸钙(COM)晶体,而在这4种体系中加入沙苑子提取液后,只形成二水草酸钙(COD)晶体,表明沙苑子提取液能抑制COM晶体生长,并且随着沙苑子提取液浓度增大,抑制作用增强。沙苑子抑制草酸钙晶体生长的可能机理进行了探讨。     

Optimized LC–ESI-ion trap MS to determine simultaneously isocorynoxeine and its phase I and II metabolites in rats with application to pharmacokinetics and mass balance investigation

Isocorynoxeine (ICOR) is one of the bioactive oxindole alkaloids in Uncaria species. This study presents the pharmacokinetics and mass balance of ICOR in rats after oral dose of 40.0 mg/kg and intravenous dose of 4.0 mg/kg through detection of ICOR and its in vivo metabolites, using an optimized LC-MS with recoveries ranged from 94 to 104%, accuracy varied from 96 to 103%, and relative standard deviation for assay being less than 5%. ICOR reached its C_max of 336.7 ng/mL in plasma 3 hr after oral dose. The phase I metabolites of ICOR, 11-hydroxyisocorynoxeine (M1), and 10-hydroxyisocorynoxeine (M2) were detected in rat heart, kidneys, urine, and feces; whereas, in rat liver and bile, M1, M2, along with phase II metabolites, 11-hydroxyisocorynoxeine 11-O-β-D-glucuronide, and 10-hydroxyisocorynoxeine 10-O-β-D-glucuronide were identified. ICOR (77.0%) was excreted into feces and urine after oral administration within 72 hr, 0.93% drained into bile in 8 hr, 17.9% biotransformed into M1 and M2 at a ratio of ca. 1:1, and 0.028% detected in main organs at t_max, in which brain uptake index is 3.2 ng/g. This work affords a developed and validated LC-MS for simultaneous determination of ICOR and its in vivo metabolites with improved precision and accuracy.     

Determination of γ-hexachlorocyclohexane and its metabolites in biological samples from rat

We have determined γ-hexachlorocyclohexane (lindane) and its metabolites in urine, serum and feces samples from rats using HPLC-UV-Vis and confirmation of mass with matrix assisted laser desorption/ ionization-time of flight (MALDI-TOF) analysis. Samples were collected from rats treated orally with lindane (17.6 mg/kg; 1/5 of LD₅₀) or vehicle for 2 weeks. Lindane and metabolites were extracted from samples with hexane and analyzed. The HPLC–MALDI-TOF is highly sensitive to the point of detecting very low level (5 ppm) of lindane and metabolites. The HPLC-UV-Vis analysis confirmed the presence of lindane in urine (386–1652 ppm), serum (207–371 ppm) and feces (5–74 ppm). Control samples had no peak corresponding to lindane. MALDI-TOF analysis of urine and serum samples showed a major peak at 293 m/z, whereas feces showed a minor peak at 292–293 m/z, which were consistent with the peak obtained for standard lindane (293 m/z). Our data indicates that HPLC-UV-Vis–MALDI-TOF combo method is sensitive for detecting and quantifying lindane and its metabolites in serum, urine and feces. Our results further showed that minor quantities of lindane and metabolites were excreted through feces confirming that the main pathway for excretion of lindane and metabolites is through urine.     

LC–MS for Identification and Elucidation of the Structure of In-Vivo and In-Vitro Metabolites of Atropine

In-vivo and in-vitro metabolism of atropine has been investigated by use of a highly specific and sensitive LC–MS ⁿ method. Feces, urine, and plasma samples were collected separately after ingestion of 25 mg kg⁻¹ atropine by healthy rats. Rat feces and urine samples were cleaned by liquid–liquid extraction and by solid-phase extraction (on C₁₈ cartridges), respectively. Methanol was added to rat plasma samples to precipitate plasma proteins. Atropine was incubated, in vitro, with homogenized liver and with intestinal flora from rats. The metabolites in the incubation solution were extracted with ethyl acetate. These pretreated samples were then analyzed by reversed-phase high-performance liquid chromatography on a C₁₈ column with methanol–ammonium acetate (2 mm, adjusted to pH 3.5 with formic acid), 70:30 (v/v), as mobile phase. Detection was by on-line MS ⁿ . Identification and elucidation of the structure of the metabolites were achieved by comparing molecular mass (ΔM), retention-times, and full-scan MS ⁿ spectra with those of the parent drug. Ten new metabolites (aponoratropine, apoatropine, hydroxymethoxyatropine, trihydroxyatropine, dimethoxyatropine, dihydroxymethoxyatropine, hydroxydimethoxyatropine, trihydroxymethoxyatropine, dihydroxydimethoxyatropine, and tropic acid) were identified in rat urine after ingestion of atropine. Nine metabolites (nortropine, tropine, aponoratropine, apoatropine, noratropine, hydroxyatropine, hydroxyatropine N-oxide, hydroxymethoxyatropine, and tropic acid) and the parent drug were detected in rat feces. Five metabolites (nortropine, tropine, tropic acid, apoatropine, and hydroxyatropine) and the parent drug were detected in rat plasma. Only two metabolites (apoatropine and noratropine) were detected in the homogenized liver incubation mixture. The hydrolyzed metabolites (tropine and tropic acid) and dehydrated metabolite apoatropine were found in the rat intestinal flora incubation mixture.     

Metabolite profiling of ginsenoside Rg1 after oral administration in rat

The goal of this study is to investigate the biotransformation of ginsenoside Rg₁ in vivo. A highly sensitive and specific LC‐MS/MS method was developed and used for metabolite identification in rat feces and urine after oral administration of ginsenoside Rg₁. Four metabolites of Rg₁ were detected in rat feces and three metabolites of Rg₁ were detected in rat urine. Deglycosylation and oxygenation were found to be the major metabolic pathways of ginsenoside Rg₁ after oral administration in rat. Except for the reported metabolites Rh₁ and protopanaxatriol, mono‐oxygenated Rg₁ and mono‐oxygenated protopanaxatriol were detected for the first time after oral administration of Rg₁. The in vivo metabolite profiling of ginsenoside Rg₁ in rat was proposed. Viewed collectively, Rg₁ was metabolized to mono‐oxygenated Rg₁, Rh₁, protopanaxatriol and the secondary metabolite mono‐oxygenated protopanaxatriol in rat. Copyright © 2014 John Wiley & Sons, Ltd.     

The in vivo absorbed constituents and metabolites of Danshen decoction in rats identified by HPLC with electrospray ionization tandem ion trap and time‐of‐flight mass spectrometry

Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is widely used for the treatment of cardiovascular and cerebrovascular diseases. This research focuses on the in vivo metabolism of Danshen decoction (DSD) in rats. After oral administration of DSD, the absorptive constituents and their metabolites in urine and plasma were analyzed by HPLC coupled with a photodiode array detector and electrospray ionization hybrid ion trap and time‐of‐flight mass spectrometry. Samples were separated on a C₁₈ column by gradient elution using 0.1% (v/v) aqueous formic acid and acetonitrile. As a result, 93 compounds from urine and 38 compounds from plasma were identified. Among them, lipo‐soluble diterpenoids (24 in urine and 15 in plasma) were reported for the first time as in vivo metabolites of DSD. According to the quantities and contents of the identified compounds, tanshinone IIA, cryptotanshinone and tanshinone I were deduced to be the major absorptive diterpenoids of DSD. Moreover, nine water‐soluble phenolics (caffeic acid, ferulic acid, danshensu, etc.) were proved to be the major absorptive constituents as reported. Most of the absorbed constituents underwent sulfation, glucuronidation, hydrogenation and hydroxylation in vivo. This investigation provided scientific evidence to obtain a more comprehensive metabolic profile of DSD. Copyright © 2014 John Wiley & Sons, Ltd.     

The Tannins from Sanguisorba officinalis L. (Rosaceae): A Systematic Study on the Metabolites of Rats Based on HPLC–LTQ–Orbitrap MS2 Analysis

Sanguisorba tannins are the major active ingredients in Sanguisorba ofJicinalis L. (Rosaceae), one of the most popular herbal medicines in China, is widely prescribed for hemostasis. In this study, three kinds of tannins extract from Sanguisorba officinalis L. (Rosaceae), and the metabolites in vivo and in vitro were detected and identified by high-pressure liquid chromatography, coupled with linear ion trap orbitrap tandem mass spectrometry (HPLC–LTQ–Orbitrap). For in vivo assessment, the rats were administered at a single dose of 150 mg/kg, after which 12 metabolites were found in urine, 6 metabolites were found in feces, and 8 metabolites were found in bile, while metabolites were barely found in plasma and tissues. For in vitro assessment, 100 μM Sanguisorba tannins were incubated with rat liver microsomes, liver cytosol, and feces, after which nine metabolites were found in intestinal microbiota and five metabolites were found in liver microsomes and liver cytosol. Moreover, the metabolic pathways of Sanguisorba tannins were proposed, which shed light on their mechanism.     

Eight New Diterpenoids from the Roots of Euphorbia nematocypha

From the dried roots of Euphorbia nematocypha, eight new diterpenoids, with ent‐atisane (i.e., 1 – 5 ) and isopimarane (i.e., 6 – 8 ) type skeletons, together with five known compounds, were isolated. The structures of these new compounds were elucidated by spectroscopic data. Compounds 1 – 8 were evaluated for their cytotoxicity against a small panel of human cancer cell lines.     

A new tigliane-type diterpene from Euphorbia dracunculoides Lam

One new tigliane-type diterpene, 4-deoxy-4(β)H-8-hydroperoxyphorbol-12-benzoate-13-isobutyrate (1), together with two known diterpenoids, 3-acetyl-5,8-dibenzoyl-14α-propanoyl-13,17-epoxy-7-myrsinaone diterpene with C9–C10 cyclised to form an additional lactone ring (2), Euphodendriane A (3) have been isolated from the whole plants of Euphorbia dracunculoides Lam. Their structures were elucidated by means of extensive spectroscopic analysis (NMR and HR-ESI-MS) and comparison with data reported in the literature. This is the first isolation of 8-hydroperoxy tigliane diterpene (1) from the genus of Euphorbia. All compounds were evaluated for their antifungal activities.     

Validated HPTLC method for the simultaneous quantification of diterpenoids in Vitex trifolia L.

Vitex trifolia L. is an important Indian medicinal plant with diverse pharmacological properties. In a recent study, we reported the isolation and antitubercular activity evaluation of three new diterpenoids from its leaves; here we have developed a validated rapid, simple, precise, and accurate high‐performance TLC method for the simultaneous quantification of isolated diterpenoids in V. trifolia. Diterpenoids, 6α,7α‐diacetoxy‐13‐hydroxy‐8(9),14‐labdadien ( A ), 13‐hydroxy‐5(10),14‐halimadien‐6‐one ( B ), and 9‐hydroxy‐13(14)‐labden‐16,15‐olide ( C ) were separated on silica gel 60F₂₅₄ high‐performance TLC plates using chloroform/acetone (98:2, v/v) as mobile phase. The quantitation of diterpenoids was carried out using densitometric reflection/absorption mode at 610 nm after postchromatographic derivatization using a vanillin/sulfuric acid reagent. A precise and accurate quantification can be performed for compounds A and B in the linear working concentration range of 333–1000 ng/band and for C in the range of 670–2000 ng/band with good correlations (r = 0.9984, 0.9991, and 0.9994, respectively). The method was validated for peak purity, precision, accuracy, robustness, LOD, and LOQ, as per the ICH guidelines. The method reported here is simple, reproducible and may be applied for the quantitative analysis of the above diterpenoids in the leaves of V. trifolia.     

The biotransformation of oleuropein in rats

A highly sensitive and specific LC‐MS/MS method was developed to investigate the in vivo bio‐transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP‐C₁₈ column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De‐glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.